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Shock (Augusta, Ga.)  2014;41(6):463-475.
Many preclinical studies in critical care medicine and related disciplines rely on hypothesis-driven research in mice. The underlying premise posits that mice sufficiently emulate numerous pathophysiological alterations produced by trauma/sepsis and can serve as an experimental platform for answering clinically relevant questions. Recently the lay press severely criticized the translational relevance of mouse models in critical care medicine. A series of provocative editorials were elicited by a highly-publicized research report in the Proceedings of the National Academy of Sciences (PNAS; February 2013), which identified an unrecognized gene expression profile mismatch between human and murine leukocytes following burn/trauma/endotoxemia. Based on their data, the authors concluded that mouse models of trauma/inflammation are unsuitable for studying corresponding human conditions. We believe this conclusion was not justified. In conjunction with resulting negative commentary in the popular press, it can seriously jeopardize future basic research in critical care medicine. We will address some limitations of that PNAS report to provide a framework for discussing its conclusions and attempt to present a balanced summary of strengths/weaknesses of use of mouse models. While many investigators agree that animal research is a central component for improved patient outcomes, it is important to acknowledge known limitations in clinical translation from mouse to man. The scientific community is responsible to discuss valid limitations without over-interpretation. Hopefully a balanced view of the strengths/weaknesses of using animals for trauma/endotoxemia/critical care research will not result in hasty discount of the clear need for using animals to advance treatment of critically ill patients.
PMCID: PMC4139038  PMID: 24569509
mouse models of critical illness; trauma; endotoxemia; sepsis; burn
2.  ‘Preconditioning’ with Low Dose Lipopolysaccharide Aggravates the Organ Injury / Dysfunction Caused by Hemorrhagic Shock in Rats 
PLoS ONE  2015;10(4):e0122096.
Male rats were ‘pretreated’ with phosphate-buffered saline (PBS; i.p.) or LPS (1 mg/kg; i.p.) 24 h prior to HS. Mean arterial pressure (MAP) was maintained at 30 ± 2 mmHg for 90 min or until 25% of the shed blood had to be re-injected to sustain MAP. This was followed by resuscitation with the remaining shed blood. Four hours after resuscitation, parameters of organ dysfunction and systemic inflammation were assessed.
HS resulted in renal dysfunction, and liver and muscular injury. At a first glance, LPS preconditioning attenuated organ dysfunction. However, we discovered that HS-rats that had been preconditioned with LPS (a) were not able to sustain a MAP at 30 mmHg for more than 50 min and (b) the volume of blood withdrawn in these animals was significantly less than in the PBS-control group. This effect was associated with an enhanced formation of the nitric oxide (NO) derived from inducible NO synthase (iNOS). Thus, a further control group in which all animals were resuscitated after 50 min of hemorrhage was performed. Then, LPS preconditioning aggravated both circulatory failure and organ dysfunction. Most notably, HS-rats pretreated with LPS exhibited a dramatic increase in NF-κB activation and pro-inflammatory cytokines.
In conclusion, LPS preconditioning predisposed animals to an earlier vascular decompensation, which may be mediated by an excess of NO production secondary to induction of iNOS and activation of NF-κB. Moreover, LPS preconditioning increased the formation of pro-inflammatory cytokines, which is likely to have contributed to the observed aggravation of organ injury/dysfunction caused by HS.
PMCID: PMC4382161  PMID: 25830444
3.  Quantification of microcirculatory blood flow: a sensitive and clinically relevant prognostic marker in murine models of sepsis 
Journal of Applied Physiology  2014;118(3):344-354.
Sepsis and sepsis-associated multiorgan failure represent the major cause of mortality in intensive care units worldwide. Cardiovascular dysfunction, a key component of sepsis pathogenesis, has received much research interest, although research translatability remains severely limited. There is a critical need for more comprehensive preclinical sepsis models, with more clinically relevant end points, such as microvascular perfusion. The purpose of this study was to compare microcirculatory blood flow measurements, using a novel application of laser speckle contrast imaging technology, with more traditional hemodynamic end points, as part of a multiparameter monitoring system in preclinical models of sepsis. Our aim, in measuring mesenteric blood flow, was to increase the prognostic sensitivity of preclinical studies. In two commonly used sepsis models (cecal ligation and puncture, and lipopolysaccharide), we demonstrate that blood pressure and cardiac output are compromised postsepsis, but subsequently stabilize over the 24-h recording period. In contrast, mesenteric blood flow continuously declines in a time-dependent manner and in parallel with the development of metabolic acidosis and organ dysfunction. Importantly, these microcirculatory perturbations are reversed by fluid resuscitation, a mainstay intervention associated with improved outcome in patients. These data suggest that global hemodynamics are maintained at the expense of the microcirculation and are, therefore, not sufficiently predictive of outcome. We demonstrate that microcirculatory blood flow is a more sensitive biomarker of sepsis syndrome progression and believe that incorporation of this biomarker into preclinical models will facilitate sophisticated proof-of-concept studies for novel sepsis interventions, providing more robust data on which to base future clinical trials.
PMCID: PMC4312846  PMID: 25477352
animal models of cardiovascular disease; microcirculation; sepsis; septic shock
4.  The challenge of translating ischemic conditioning from animal models to humans: the role of comorbidities 
Disease Models & Mechanisms  2014;7(12):1321-1333.
Following a period of ischemia (local restriction of blood supply to a tissue), the restoration of blood supply to the affected area causes significant tissue damage. This is known as ischemia-reperfusion injury (IRI) and is a central pathological mechanism contributing to many common disease states. The medical complications caused by IRI in individuals with cerebrovascular or heart disease are a leading cause of death in developed countries. IRI is also of crucial importance in fields as diverse as solid organ transplantation, acute kidney injury and following major surgery, where post-operative organ dysfunction is a major cause of morbidity and mortality. Given its clinical impact, novel interventions are urgently needed to minimize the effects of IRI, not least to save lives but also to reduce healthcare costs. In this Review, we examine the experimental technique of ischemic conditioning, which entails exposing organs or tissues to brief sub-lethal episodes of ischemia and reperfusion, before, during or after a lethal ischemic insult. This approach has been found to confer profound tissue protection against IRI. We discuss the translation of ischemic conditioning strategies from bench to bedside, and highlight where transition into human clinical studies has been less successful than in animal models, reviewing potential reasons for this. We explore the challenges that preclude more extensive clinical translation of these strategies and emphasize the role that underlying comorbidities have in altering the efficacy of these strategies in improving patient outcomes.
PMCID: PMC4257001  PMID: 25481012
Comorbidities; Ischemic postconditioning; Ischemic preconditioning; Remote ischemic preconditioning
5.  Gender Dimorphism of the Cardiac Dysfunction in Murine Sepsis: Signalling Mechanisms and Age-Dependency 
PLoS ONE  2014;9(6):e100631.
Development of cardiac dysfunction is associated with increased morbidity and mortality in patients with sepsis. Increasing evidence shows that gender determines the degree of inflammatory response of the host and that females tolerate sepsis better than males. It is unknown whether gender affects the cardiac dysfunction in animals or patients with sepsis. To investigate this, male or female C57BL/6 mice were subjected to either lipopolysaccharide (LPS)/peptidoglycan (PepG) co-administration or cecal ligation and puncture (CLP). At 18 hours after LPS/PepG injection or 24 hours after CLP, cardiac function was evaluated by echocardiography. The septic insult caused a significant cardiac dysfunction in both genders. However, the cardiac dysfunction was significantly less pronounced in females in comparison with males subjected to LPS (3 mg/kg)/PepG (0.1 mg/kg) or CLP. Compared with males injected with LPS (3 mg/kg)/PepG (0.1 mg/kg), western blotting analysis of the myocardium from females injected with LPS/PepG revealed i) profound increases in phosphorylation of Akt and eNOS; ii) significant decreases in phosphorylation of IκBα, nuclear translocation of the NF-κB subunit p65, decreased expression of iNOS and decreased synthesis of TNF-α and IL-6 in the heart. However, the gender dimorphism of the cardiac dysfunction secondary to LPS/PepG was not observed when higher doses of LPS (9 mg/kg)/PepG (1 mg/kg) were used. In conclusion, the cardiac dysfunction caused by sepsis was less pronounced in female than in male mice. The protection of female hearts against the dysfunction associated with sepsis is (at least in part) attributable to cardiac activation of the Akt/eNOS survival pathway, decreased activation of NF-κB, and decreased expression of iNOS, TNF-α and IL-6. It should be noted that the observed gender dimorphism of the cardiac dysfunction in sepsis was not seen when a very severe stimulus (high dose of LPS/PepG co-administration) was used to cause cardiac dysfunction.
PMCID: PMC4063956  PMID: 24945834
6.  Erythropoietin in the critically ill: do we ask the right questions? 
Critical Care  2012;16(5):319.
There is a plethora of experimental data on the potential therapeutic benefits of recombinant human erythropoietin (rhEPO) and its synthetic derivatives in critical care medicine, in particular in ischemia/reperfusion injury. Most of the recent clinical trials have not shown clear benefits, and, in some patients, EPO-aggravated morbidity and mortality was even reported. Treatment with rhEPO has been successfully used in patients with anemia resulting from chronic kidney disease, but even a subset of this patient population does not adequately respond to rhEPO therapy. The following viewpoint uses rhEPO as an example to highlight the possible pitfalls in current practice using young healthy animals for the evaluation of therapies to treat patients of variable age and underlying chronic co-morbidity.
PMCID: PMC3682241  PMID: 23016869
7.  Bench-to-bedside review: Erythropoietin and its derivatives as therapies in critical care 
Critical Care  2012;16(4):229.
Erythropoietin (EPO) is known to have numerous biological functions. Its primary function in the body is to increase red blood cell numbers by way of preventing the apoptosis of erythroid progenitor cells via the homodimeric EPO receptor. The discovery that the local production of EPO within the brain in response to hypoxia or ischemia protects neurons against injury via an anti-apoptotic effect formed the basis of the hypothesis that the local generation of EPO limits the extent of injury. Although the hypothesis proved to be true in pre-clinical models of ischemia/reperfusion injury and inflammation, the randomized, controlled clinical trials that followed demonstrated serious adverse events of EPO due to activation of the hematopoietic system. Consequently, derivatives of EPO that lacked erythropoietic activity were discovered to reduce injury in many pre-clinical models associated with ischemia and inflammation. Unfortunately, there are no published clinical trials to determine the efficacy of non-erythropoietic derivatives of EPO in humans.
PMCID: PMC3580677  PMID: 22839413
8.  Erythropoietin attenuates cardiac dysfunction in experimental sepsis in mice via activation of the β-common receptor 
Disease Models & Mechanisms  2013;6(4):1021-1030.
There is limited evidence that the tissue-protective effects of erythropoietin are mediated by a heterocomplex of the erythropoietin receptor and the β-common receptor (‘tissue-protective receptor’), which is pharmacologically distinct from the ‘classical’ erythropoietin receptor homodimer that is responsible for erythropoiesis. However, the role of the β-common receptor and/or erythropoietin in sepsis-induced cardiac dysfunction (a well known, serious complication of sepsis) is unknown. Here we report for the first time that the β-common receptor is essential for the improvements in the impaired systolic contractility afforded by erythropoietin in experimental sepsis. Cardiac function was assessed in vivo (echocardiography) and ex vivo (Langendorff-perfused heart) in wild-type and β-common receptor knockout mice, that were subjected to lipopolysaccharide (9 mg/kg body weight; young mice) for 16–18 hours or cecal ligation and puncture (aged mice) for 24 hours. Mice received erythropoietin (1000 IU/kg body weight) 1 hour after lipopolysaccharide or cecal ligation and puncture. Erythropoietin reduced the impaired systolic contractility (in vivo and ex vivo) caused by endotoxemia or sepsis in young as well as old wild-type mice in a β-common-receptor-dependent fashion. Activation by erythropoietin of the β-common receptor also resulted in the activation of well-known survival pathways (Akt and endothelial nitric oxide synthase) and inhibition of pro-inflammatory pathways (glycogen synthase kinase-3β, nuclear factor-κB and interleukin-1β). All the above pleiotropic effects of erythropoietin were lost in β-common receptor knockout mice. Erythropoietin attenuates the impaired systolic contractility associated with sepsis by activation of the β-common receptor, which, in turn, results in activation of survival pathways and inhibition of inflammation.
PMCID: PMC3701221  PMID: 23519033
9.  Inhibition of IκB kinase reduces the multiple organ dysfunction caused by sepsis in the mouse 
Disease Models & Mechanisms  2013;6(4):1031-1042.
Nuclear factor κB (NF-κB) plays a pivotal role in sepsis. Activation of NF-κB is initiated by the signal-induced ubiquitylation and subsequent degradation of inhibitors of kappa B (IκBs) primarily via activation of the IκB kinase (IKK). This study was designed to investigate the effects of IKK inhibition on sepsis-associated multiple organ dysfunction and/or injury (MOD) and to elucidate underlying signaling mechanisms in two different in vivo models: male C57BL/6 mice were subjected to either bacterial cell wall components [lipopolysaccharide and peptidoglycan (LPS/PepG)] or underwent cecal ligation and puncture (CLP) to induce sepsis-associated MOD. At 1 hour after LPS/PepG or CLP, mice were treated with the IKK inhibitor IKK 16 (1 mg/kg body weight). At 24 hours, parameters of organ dysfunction and/or injury were assessed in both models. Mice developed a significant impairment in systolic contractility (echocardiography), and significant increases in serum creatinine, serum alanine aminotransferase and lung myeloperoxidase activity, thus indicating cardiac dysfunction, renal dysfunction, hepatocellular injury and lung inflammation, respectively. Treatment with IKK 16 attenuated the impairment in systolic contractility, renal dysfunction, hepatocellular injury and lung inflammation in LPS/PepG-induced MOD and in polymicrobial sepsis. Compared with mice that were injected with LPS/PepG or underwent CLP, immunoblot analyses of heart and liver tissues from mice that were injected with LPS/PepG or underwent CLP and were also treated with IKK 16 revealed: (1) significant attenuation of the increased phosphorylation of IκBα; (2) significant attenuation of the increased nuclear translocation of the NF-κB subunit p65; (3) significant attenuation of the increase in inducible nitric oxide synthase (iNOS) expression; and (4) a significant increase in the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Here, we report for the first time that delayed IKK inhibition reduces MOD in experimental sepsis. We suggest that this protective effect is (at least in part) attributable to inhibition of inflammation through NF-κB, the subsequent decrease in iNOS expression and the activation of the Akt-eNOS survival pathway.
PMCID: PMC3701222  PMID: 23649820
10.  Pharmacological preconditioning with erythropoietin attenuates the organ injury and dysfunction induced in a rat model of hemorrhagic shock 
Disease Models & Mechanisms  2012;6(3):701-709.
Pre-treatment with erythropoietin (EPO) has been demonstrated to exert tissue-protective effects against ‘ischemia-reperfusion’-type injuries. This protection might be mediated by mobilization of bone marrow endothelial progenitor cells (EPCs), which are thought to secrete paracrine factors. These effects could be exploited to protect against tissue injury induced in cases where hemorrhage is foreseeable, for example, prior to major surgery. Here, we investigate the effects of EPO pre-treatment on the organ injury and dysfunction induced by hemorrhagic shock (HS). Recombinant human EPO (1000 IU/kg/day i.p.) was administered to rats for 3 days. Rats were subjected to HS on day 4 (pre-treatment protocol). Mean arterial pressure was reduced to 35±5 mmHg for 90 minutes, followed by resuscitation with 20 ml/kg Ringer’s lactate for 10 minutes and 50% of the shed blood for 50 minutes. Rats were sacrificed 4 hours after the onset of resuscitation. EPC (CD34+/flk-1+ cell) mobilization was measured following the 3-day pre-treatment with EPO and was significantly increased compared with rats pre-treated with phosphate-buffered saline. EPO pre-treatment significantly attenuated organ injury and dysfunction (renal, hepatic and neuromuscular) caused by HS. In livers from rats subjected to HS, EPO enhanced the phosphorylation of Akt (activation), glycogen synthase kinase-3β (GSK-3β; inhibition) and endothelial nitric oxide synthase (eNOS; activation). In the liver, HS also caused an increase in nuclear translocation of p65 (activation of NF-κB), which was attenuated by EPO. This data suggests that repetitive dosing with EPO prior to injury might protect against the organ injury and dysfunction induced by HS, by a mechanism that might involve mobilization of CD34+/flk-1+ cells, resulting in the activation of the Akt-eNOS survival pathway and inhibition of activation of GSK-3β and NF-κB.
PMCID: PMC3634653  PMID: 23264564
11.  Dopexamine can attenuate the inflammatory response and protect against organ injury in the absence of significant effects on hemodynamics or regional microvascular flow 
Critical Care  2013;17(2):R57.
The effects of dopexamine, a β2-agonist, on perioperative and sepsis-related hemodynamic, microvascular, immune, and organ dysfunction are controversial and poorly understood. We investigated these effects in a rodent model of laparotomy and endotoxemia.
In two experiments, 80 male Wistar rats underwent laparotomy. In 64 rats, this was followed by administration of endotoxin; the remainder (16) underwent sham endotoxemia. Endotoxemic animals received either dopexamine at 0.5, 1, or 2 μg/kg/min or 0.9% saline vehicle (controls) as resuscitation fluid. The effects of dopexamine on global hemodynamics, mesenteric regional microvascular flow, renal and hepatic function and immune activation were evaluated.
Endotoxin administration was associated with a systemic inflammatory response (increased plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10, as well as cell-adhesion molecules CD11a and CD11b), and increased pulmonary myeloperoxidase (MPO) activity (indicating pulmonary leukocyte infiltration), whereas biochemical changes demonstrated lactic acidosis with significant renal and hepatic injury. Dopexamine administration was associated with less-severe lactic acidosis (pooled dopexamine versus controls, (lactate, 2.2 mM ± 0.2 mM versus 4.0 mM ± 0.5 mM; P < 0.001) and reductions in the systemic inflammatory response (pooled dopexamine versus control, 4 hour (TNF-α): 324 pg/ml ± 93 pg/ml versus 97 pg/ml ± 14 pg/ml, p < 0.01), pulmonary myeloperoxidase (MPO) activity, and hepatic and renal injury (pooled dopexamine versus control (ALT): 81 IU/L ± 4 IU/L versus 138 IU/L ± 25 IU/L; P < 0.05; (creatinine): 49.4 μM ± 3.9 μM versus 76.2 μM ± 9.8 μM; P < 0.005). However, in this study, clinically relevant doses of dopexamine were not associated with clinically significant changes in MAP, CI, or gut regional microvascular flow.
In this model, dopexamine can attenuate the systemic inflammatory response, reduce tissue leukocyte infiltration, and protect against organ injury at doses that do not alter global hemodynamics or regional microvascular flow. These findings suggest that immunomodulatory effects of catecholamines may be clinically significant when used in critically ill surgical patients and are independent of their hemodynamic actions.
PMCID: PMC3672538  PMID: 23531318
12.  Enhanced IL-17 signalling following myocardial ischaemia/reperfusion injury 
International Journal of Cardiology  2013;163(3):326-334.
IL-17A and IL-17F are pro-inflammatory cytokines which induce the expression of several cytokines, chemokines and matrix metalloproteinases (MMPs) in target cells. IL-17 cytokines have recently attracted huge interest due to their pathogenic role in diseases such as arthritis and inflammatory bowel disease although a role for IL-17 cytokines in myocardial infarction (MI) has not previously been described.
In vivo MI was performed by coronary artery occlusion in the absence or presence of a neutralizing IL-17 antibody for blocking IL-17 actions in vivo. IL-17 signaling was also assessed in isolated primary cardiomyocytes by Western blot, mRNA expression and immunostaining.
Expression of IL-17A, IL-17F and the IL-17 receptor (IL-17RA) were all increased following MI. Expression of several IL-17 target genes, including Cxcl1, Cxcl2, IL-1β, iNOS and IL-6 was also upregulated following MI. In addition, IL-17A promoted the expression of Cxcl1 and IL-6 in isolated cardiomyocytes in a MAPK and PI(3)K-dependent manner. IL-17A and ischaemia/reperfusion (I/R) injury were found to have an additive effect on Cxcl1 expression, suggesting that IL-17 may enhance myocardial neutrophil recruitment during MI. Moreover, protein levels of both IL-17R and IL-17A were enhanced following in vivo MI. Finally, blocking IL-17 signaling in vivo reduced the levels of apoptotic cell death markers following in vivo MI.
These data imply that the expression of IL-17 cytokines and their receptor are elevated during myocardial I/R injury and may play a fundamental role in post infarct inflammatory and apoptotic responses.
PMCID: PMC3581775  PMID: 22030025
IL-17; Myocardial; Ischaemia/reperfusion; IL-17 receptor; Cxcl1; MAPK
13.  Delayed Administration of Pyroglutamate Helix B Surface Peptide (pHBSP), a Novel Nonerythropoietic Analog of Erythropoietin, Attenuates Acute Kidney Injury 
Molecular Medicine  2012;18(1):719-727.
In preclinical studies, erythropoietin (EPO) reduces ischemia-reperfusion–associated tissue injury (for example, stroke, myocardial infarction, acute kidney injury, hemorrhagic shock and liver ischemia). It has been proposed that the erythropoietic effects of EPO are mediated by the classic EPO receptor homodimer, whereas the tissue-protective effects are mediated by a hetero-complex between the EPO receptor monomer and the β-common receptor (termed “tissue-protective receptor”). Here, we investigate the effects of a novel, selective-ligand of the tissue-protective receptor (pyroglutamate helix B surface peptide [pHBSP]) in a rodent model of acute kidney injury/dysfunction. Administration of pHBSP (10 μg/kg intraperitoneally [i.p.] 6 h into reperfusion) or EPO (1,000 IU/kg i.p. 4 h into reperfusion) to rats subjected to 30 min ischemia and 48 h reperfusion resulted in significant attenuation of renal and tubular dysfunction. Both pHBSP and EPO enhanced the phosphorylation of Akt (activation) and glycogen synthase kinase 3β (inhibition) in the rat kidney after ischemia-reperfusion, resulting in prevention of the activation of nuclear factor-κB (reduction in nuclear translocation of p65). Interestingly, the phosphorylation of endothelial nitric oxide synthase was enhanced by EPO and, to a much lesser extent, by pHBSP, suggesting that the signaling pathways activated by EPO and pHBSP may not be identical.
PMCID: PMC3388125  PMID: 22415011
14.  Erythropoietin in the intensive care unit: beyond treatment of anemia 
Erythropoietin (EPO) is the major hormone stimulating the production and differentiation of red blood cells. EPO is used widely for treating anemia of critical illness or anemia induced by chemotherapy. EPO at pharmacological doses is used in this setting to raise hemoglobin levels (by preventing the apoptosis of erythroid progenitor cells) and is designed to reduce patient exposure to allogenic blood through transfusions. Stroke, heart failure, and acute kidney injury are a frequently encountered clinical problem. Unfortunately, in the intensive care unit advances in supportive interventions have done little to reduce the high mortality associated with these conditions. Tissue protection with EPO at high, nonpharmacological doses after injury has been found in the brain, heart, and kidney of several animal models. It is now well known that EPO has anti-apoptotic effects in cells other than erythroid progenitor cells, which is considered to be independent of EPOs erythropoietic activities. This review article summarizes what is known in preclinical models of critical illness and discusses why this does not correlate with randomized, controlled clinical trials.
PMCID: PMC3224459  PMID: 21943500
15.  A Nonerythropoietic Peptide that Mimics the 3D Structure of Erythropoietin Reduces Organ Injury/Dysfunction and Inflammation in Experimental Hemorrhagic Shock 
Molecular Medicine  2011;17(9-10):883-892.
Recent studies have shown that erythropoietin, critical for the differentiation and survival of erythrocytes, has cytoprotective effects in a wide variety of tissues, including the kidney and lung. However, erythropoietin has been shown to have a serious side effect—an increase in thrombovascular effects. We investigated whether pyroglutamate helix B-surface peptide (pHBSP), a nonerythropoietic tissue-protective peptide mimicking the 3D structure of erythropoietin, protects against the organ injury/ dysfunction and inflammation in rats subjected to severe hemorrhagic shock (HS). Mean arterial blood pressure was reduced to 35 ± 5 mmHg for 90 min followed by resuscitation with 20 mL/kg Ringer Lactate for 10 min and 50% of the shed blood for 50 min. Rats were euthanized 4 h after the onset of resuscitation. pHBSP was administered 30 min or 60 min into resuscitation. HS resulted in significant organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung). In rats subjected to HS, pHBSP significantly attenuated (i) organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung), (ii) increased the phosphorylation of Akt, glycogen synthase kinase-3β and endothelial nitric oxide synthase, (iii) attenuated the activation of nuclear factor (NF)-κB and (iv) attenuated the increase in p38 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. pHBSP protects against multiple organ injury/dysfunction and inflammation caused by severe hemorrhagic shock by a mechanism that may involve activation of Akt and endothelial nitric oxide synthase, and inhibition of glycogen synthase kinase-3β and NF-κB.
PMCID: PMC3188881  PMID: 21607291
16.  Pioglitazone improves lipid and insulin levels in overweight rats on a high cholesterol and fructose diet by decreasing hepatic inflammation 
British Journal of Pharmacology  2010;160(8):1892-1902.
Background and purpose:
Nutrient overload leads to obesity and insulin resistance. Pioglitazone, a selective peroxisome proliferator-activated receptor (PPAR)γ agonist, is currently used to manage insulin resistance, but the specific molecular mechanisms activated by PPARγ are not yet fully understood. Recent studies suggest the involvement of suppressor of cytokine signalling (SOCS)-3 in the pathogenesis of insulin resistance. This study aimed to investigate the hepatic signalling pathway activated by PPARγ activation in a non-genetic insulin-resistant animal model.
Experimental approach:
Male Wistar rats were maintained on a high-cholesterol fructose (HCF) diet for 15 weeks. Pioglitazone (3 mg·kg−1) was administered orally for the last 4 weeks of this diet. At the end of the treatment, serum was collected for biochemical analysis. Levels of PPARγ, SOCS-3, pro-inflammatory markers, insulin receptor substrate-2 and Akt/glycogen synthase kinase-3β phosphorylation were assesed in rat liver.
Key results:
Rats fed the HCF diet exhibited hyperlipidemia, hyperinsulinemia, impaired glucose tolerance and insulin resistance. Pioglitazone administration evoked a significant improvement in lipid metabolism and insulin responsiveness. This was accompanied by reduced hepatic expression of SOCS-3, interleukin-6, tumour necrosis factor-α and markers of neutrophil infiltration. Diet-induced PPARγ expression was unaffected by the pioglitazone treatment.
Conclusion and implications:
Chronic pioglitazone administration reduced hepatic inflammatory responses in rats fed a HCF diet. These effects were associated with changes in hepatic expression of SOCS-3, which may be a crucial link between the reduced local inflammation and the improved insulin signalling.
This article is commented on by Chatterjee, pp. 1889–1891 of this issue. To view this commentary visit
PMCID: PMC2958635  PMID: 20233219
pioglitazone; high cholesterol and fructose diet; insulin resistance; inflammation; PPARγ; SOCS-3
17.  New targets of urocortin-mediated cardioprotection 
The urocortin (UCN) hormones UCN1 and UCN2 have been shown previously to confer significant protection against myocardial ischaemia/reperfusion (I/R) injury; however, the molecular mechanisms underlying their action are poorly understood. To further define the transcriptional effect of UCNs that underpins their cardioprotective activity, a microarray analysis was carried out using an in vivo rat coronary occlusion model of I/R injury. Infusion of UCN1 or UCN2 before the onset of reperfusion resulted in the differential regulation of 66 and 141 genes respectively, the majority of which have not been described previously. Functional analysis demonstrated that UCN-regulated genes are involved in a wide range of biological responses, including cell death (e.g. X-linked inhibitor of apoptosis protein), oxidative stress (e.g. nuclear factor erythroid derived 2-related factor 1/nuclear factor erythroid derived 2-like 1) and metabolism (e.g. Prkaa2/AMPK). In addition, both UCN1 and UCN2 were found to modulate the expression of a host of genes involved in G-protein-coupled receptor (GPCR) signalling including Rac2, Gnb1, Dab2ip (AIP1), Ralgds, Rnd3, Rap1a and PKA, thereby revealing previously unrecognised signalling intermediates downstream of CRH receptors. Moreover, several of these GPCR-related genes have been shown previously to be involved in mitogen-activated protein kinase (MAPK) activation, suggesting a link between CRH receptors and induction of MAPKs. In addition, we have shown that both UCN1 and UCN2 significantly reduce free radical damage following myocardial infarction, and comparison of the UCN gene signatures with that of the anti-oxidant tempol revealed a significant overlap. These data uncover novel gene expression changes induced by UCNs, which will serve as a platform to further understand their mechanism of action in normal physiology and cardioprotection.
PMCID: PMC3069736  PMID: 20501665
18.  Junctional adhesion molecule (JAM)-C mediates leukocyte infiltration in response to ischemia reperfusion injury 
JAM-C is an adhesion molecule that has multiple roles in inflammation and vascular biology but many aspects of its functions under pathological conditions are unknown. Here we investigated the role of JAM-C in leukocyte migration in response to ischemia reperfusion (I/R) injury.
Methods and Results
Pre-treatment of mice with soluble JAM-C (sJAM-C), used as a pharmacological blocker of JAM-C-mediated reactions, significantly suppressed leukocyte migration in models of kidney and cremaster muscle I/R injury (39 and 51% inhibition, respectively). Furthermore, in the cremaster muscle model (studied by intravital microscopy), both leukocyte adhesion and transmigration were suppressed in JAM-C deficient mice (JAM-C−/−) and enhanced in mice over-expressing JAM-C in their endothelial cells (ECs). Analysis of JAM-C subcellular expression by immunoelectron microscopy indicated that in I/R-injured tissues, EC JAM-C was redistributed from cytoplasmic vesicles and EC junctional sites to non-junctional plasma membranes, a response that may account for the role of JAM-C in both leukocyte adhesion and transmigration under conditions of I/R injury.
The findings demonstrate a role for EC JAM-C in mediating leukocyte adhesion and transmigration in response to I/R injury and indicate the existence of a novel regulatory mechanism for redistribution and hence function of EC JAM-C in vivo.
PMCID: PMC2746810  PMID: 19574560
JAM-C; Ischemia reperfusion injury; Leukocyte transmigration; Inflammation; Adhesion molecules
19.  Role of PPAR-δ in the development of zymosan-induced multiple organ failure: an experiment mice study 
Peroxisome proliferator-activated receptor (PPAR)-beta/delta is a nuclear receptor transcription factor that regulates gene expression in many important biological processes. It is expressed ubiquitously, especially white adipose tissue, heart, muscle, intestine, placenta and macrophages but many of its functions are unknown. Saturated and polyunsaturated fatty acids activate PPAR-beta/delta, but physiological ligands have not yet been identified. In the present study, we investigated the anti-inflammatory effects of PPAR-beta/delta activation, through the use of GW0742 (0,3 mg/kg 10% Dimethyl sulfoxide (DMSO) i.p), a synthetic high affinity ligand, on the development of zymosan-induced multiple organ failure (MOF).
Multiple organ failure (MOF) was induced in mice by administration of zymosan (given at 500 mg/kg, i.p. as a suspension in saline). The control groups were treated with vehicle (0.25 ml/mouse saline), while the pharmacological treatment was the administration of GW0742 (0,3 mg/kg 10% DMSO i.p. 1 h and 6 h after zymosan administration). MOF and systemic inflammation in mice was assessed 18 hours after administration of zymosan.
Treatment with GW0742 caused a significant reduction of the peritoneal exudate formation and of the neutrophil infiltration caused by zymosan resulting in a reduction in myeloperoxidase activity. The PPAR-beta/delta agonist, GW0742, at the dose of 0,3 mg/kg in 10% DMSO, also attenuated the multiple organ dysfunction syndrome caused by zymosan. In pancreas, lung and gut, immunohistochemical analysis of some end points of the inflammatory response, such as inducible nitric oxide synthase (iNOS), nitrotyrosine, poly (ADP-ribose) (PAR), TNF- and IL-1as well as FasL, Bax, Bcl-2 and apoptosis, revealed positive staining in sections of tissue obtained from zymosan-injected mice. On the contrary, these parameters were markedly reduced in samples obtained from mice treated with GW0742
In this study, we have shown that GW0742 attenuates the degree of zymosan-induced non-septic shock in mice.
PMCID: PMC2844385  PMID: 20167109
20.  Insulin Reduces Cerebral Ischemia/Reperfusion Injury in the Hippocampus of Diabetic Rats 
Diabetes  2009;58(1):235-242.
OBJECTIVE—There is evidence that insulin reduces brain injury evoked by ischemia/reperfusion (I/R). However, the molecular mechanisms underlying the protective effects of insulin remain unknown. Insulin is a well-known inhibitor of glycogen synthase kinase-3β (GSK-3β). Here, we investigate the role of GSK-3β inhibition on I/R-induced cerebral injury in a rat model of insulinopenic diabetes.
RESEARCH DESIGN AND METHODS—Rats with streptozotocin-induced diabetes were subjected to 30-min occlusion of common carotid arteries followed by 1 or 24 h of reperfusion. Insulin (2–12 IU/kg i.v.) or the selective GSK-3β inhibitor TDZD-8 (0.2–3 mg/kg i.v.) was administered during reperfusion.
RESULTS—Insulin or TDZD-8 dramatically reduced infarct volume and levels of S100B protein, a marker of cerebral injury. Both drugs induced phosphorylation of the Ser9 residue, thereby inactivating GSK-3β in the rat hippocampus. Insulin, but not TDZD-8, lowered blood glucose. The hippocampi of the drug-treated animals displayed reduced oxidative stress at 1 h of reperfusion as shown by the decreased generation of reactive oxygen species and lipid peroxidation. I/R-induced activation of nuclear factor-κB was attenuated by both drug treatments. At 24 h of reperfusion, TDZD-8 and insulin significantly reduced plasma levels of tumor necrosis factor-α; neutrophil infiltration, measured as myeloperoxidase activity and intercellular-adhesion-molecule-1 expression; and cyclooxygenase-2 and inducible-NO-synthase expression.
CONCLUSIONS—Acute administration of insulin or TDZD-8 reduced cerebral I/R injury in diabetic rats. We propose that the inhibitory effect on the activity of GSK-3β contributes to the protective effect of insulin independently of any effects on blood glucose.
PMCID: PMC2606878  PMID: 18840784
21.  Lysophosphatidylcholine reduces the organ injury and dysfunction in rodent models of Gram-negative and Gram-positive shock 
British Journal of Pharmacology  2006;148(6):769-777.
Lysophosphatidylcholine (LPC) modulates the inflammatory response and reduces mortality in animal models of sepsis. Here, we investigate the effects of LPC from synthetic (sLPC) and natural, soy bean derived LPC, (nLPC) sources on the organ injury/dysfunction caused by systemic administration of lipopolysaccharide (LPS) or peptidoglycan (PepG) and lipoteichoic acid (LTA).Rats were subjected to (i) endotoxaemia (LPS 6 mg kg–1 i.v.) and treated with sLPC (1–100 mg kg−1), (ii) endotoxaemia and treated with nLPC (10 mg kg−1) or (iii) Gram-positive shock (PepG 10 mg kg–1 and LTA 3 mg kg–1 i.v.) and treated with sLPC (10 mg kg−1).Endotoxaemia or Gram-positive shock for 6 h resulted in increases in serum makers of renal dysfunction and liver, pancreatic and neuromuscular injury.Administration of sLPC, at 1 or 2 h after LPS, dose dependently (1–10 mg kg−1) reduced the organ injury/dysfunction. High doses of sLPC (30 and 100 mg kg−1) were shown to be detrimental in endotoxaemia. sLPC also afforded protection against the organ injury/dysfunction caused by Gram-positive shock. nLPC was found to be protective in endotoxaemic animals.The beneficial effects of sLPC were associated with an attenuation in circulating levels of interleukin-1β (IL-1β).In conclusion, LPC dose and time dependently reduces the organ injury and circulating IL-1β levels caused by Gram-negative or Gram-positive shock in the rat. Thus, we speculate that appropriate doses of LPC may be useful in reducing the degree of organ injury and dysfunction associated with shock of various aetiologies.
PMCID: PMC1617069  PMID: 16751791
Endotoxin; lipoteichoic acid; lysophosphatidylcholine; LPC; LPS; oxidised lipoprotein; peptidoglycan; sepsis; shock
22.  Beneficial effects of erythropoietin in preclinical models of shock and organ failure 
Critical Care  2007;11(3):132.
Erythropoietin protects many organs against the tissue injury and dysfunction caused by ischaemia/reperfusion and excessive inflammation. This editorial comment discusses the effects of erythropoietin in preclinical models of septic shock, endotoxemia, hemorrhagic shock, spinal cord trauma and zymosan-induced multiple organ failure.
PMCID: PMC2206404  PMID: 17543134
23.  Reduction of experimental colitis in the rat by inhibitors of glycogen synthase kinase-3β 
British Journal of Pharmacology  2005;147(5):575-582.
The effects of the inhibitors of glycogen synthase kinase-3β (GSK-3β), TDZD-8 and SB 415286, which can substantially reduce the systemic inflammation associated with endotoxic shock in vivo, have now been investigated on the acute colitis provoked by trinitrobenzene sulphonic acid (TNBS) in the rat.Administration of the GSK-3β inhibitor TDZD-8 (0.1, 0.33 or 1.0 mg kg−1, s.c., b.i.d., for 3 days) caused a dose-dependent reduction in the colonic inflammation induced by intracolonic TNBS assessed after 3 days, both as the area of macroscopic involvement and as a score using 0–10 scale.Likewise, following administration of the GSK-3β inhibitor SB 415286 (0.1, 0.33 or 1.0 mg kg−1, s.c., b.i.d., for 3 days), the extent and degree of the TNBS-provoked colonic inflammation was reduced.Administration of either TDZD-8 or SB 415286 reduced the fall in body weight following challenge with TNBS at each dose level studied.The increase in myeloperoxidase activity, an index of neutrophil infiltration into the TNBS-induced inflamed colon, was significantly inhibited by both TDZD-8 and SB 415286 at each dose level.The increase in the levels of the proinflammatory cytokine, TNF-α, in the inflamed colon was also significantly inhibited by either compound at the highest doses evaluated.The elevated levels of the transcription factor NF-κB subunit p65, as determined by Western blot in the nuclear extracts from the TNBS-provoked inflamed colonic tissue, were dose-dependently reduced by TDZD-8 or SB 415286 treatment.These findings demonstrate that two chemically distinct selective inhibitors of the activity of GSK-3β reduce the inflammation and tissue injury in a rat model of acute colitis. The mechanisms underlying this anti-inflammatory action may be related to downregulation of NF-κB activity, involved in the generation of proinflammatory mediators.
PMCID: PMC1616977  PMID: 16314851
Glycogen synthase kinase-3β; GSK-3β inhibitors; NF-κB; NF-κB p65; SB 415286; TDZD-8; TNBS colitis; TNF-α
24.  Inhibition of endogenous hydrogen sulfide formation reduces the organ injury caused by endotoxemia 
British Journal of Pharmacology  2005;146(4):498-505.
Hydrogen sulfide (H2S) is a naturally occurring gaseous transmitter, which may play important roles in normal physiology and disease. Here, we investigated the role of H2S in the organ injury caused by severe endotoxemia in the rat.Male Wistar rats were subjected to acute endotoxemia (Escherichia coli lipopolysaccharide (LPS) 6 mg kg−1 intravenously (i.v.) for 6 h) and treated with vehicle (saline, 1 ml kg−1 i.v.) or DL-propargylglycine (PAG, 10–100 mg kg−1 i.v.), an inhibitor of the H2S-synthesizing enzyme cystathionine-γ-lyase (CSE). PAG was administered either 30 min prior to or 60 min after the induction of endotoxemia.Endotoxemia resulted in circulatory failure (hypotension and tachycardia) and an increase in serum levels of alanine aminotransferase and aspartate aminotransferase (markers for hepatic injury), lipase (indicator of pancreatic injury) and creatine kinase (indicator of neuromuscular injury). In the liver, endotoxemia induced a significant increase in the myeloperoxidase (MPO) activity, and in the expression and activity of the H2S-synthesizing enzymes CSE and cystathionine-β-synthase.Administration of PAG either prior to or after the injection of LPS dose-dependently reduced the hepatocellular, pancreatic and neuromuscular injury caused by endotoxemia, but not the circulatory failure. Pretreatment of rats with PAG abolished the LPS-induced increase in the MPO activity and in the formation of H2S and in the liver.These findings support the view that an enhanced formation of H2S contributes to the pathophysiology of the organ injury in endotoxemia. We propose that inhibition of H2S synthesis may be a useful therapeutic strategy against the organ injury associated with sepsis and shock.
PMCID: PMC1751176  PMID: 16100527
Hydrogen sulfide; DL-propargylglycine; LPS; cystathionine-γ-lyase; CSE; rat
25.  Ligands of the peroxisome proliferator-activated receptor-γ and heart failure 
British Journal of Pharmacology  2003;142(6):1049-1051.
This Commentary originally appeared in the British Journal of Pharmacology in January 2004 (Br J Pharmacol 141 (1), 1–3). That version, regrettably, contained a number of errors and the corrected version is printed here, as an Erratum.
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid and thyroid hormone receptors. The PPAR subfamily comprises of three members, PPAR-α, PPAR-β and PPAR-γ. There is good evidence that ligands of PPAR-γ, including certain thiazolinediones, reduce myocardial tissue injury and infarct size. The use of PPAR-γ agonists in the treatment of heart failure is, however, controversial.
PMCID: PMC1575122  PMID: 15303252
PPAR; nuclear receptors; myocardial infarction; myocardial ischaemia

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